The ultimate goal of this research project is to develop osteoinductive osteoblast/polymer constructs for bone regeneration and repair. We will investigate the effects of flow and fluid shear, as well as scaffold pore architecture and surface composition on the proliferation and osteogenic differentiation of marrow stromal cells seeded in three-dimensional biodegradable polymer scaffolds and cultured in vitro in a flow perfusion bioreactor developed in our laboratory. We will determine the effects of these parameters on the secretion and deposition of osteoinductive and angiogenic growth factors, such as bone morphogenetic protein-2 and vascular endothelial growth factor, the secretion of bone matrix proteins, such as osteocalcin, and the deposition of mineralized extracellular matrix. We will also investigate the effects of fluid shear and in vitro culture period on mineralized tissue formation after implantation of the cell/polymer constructs in the mesentery of rats and will elucidate the contributions of the mineralized extracellular matrix generated in vitro and the differentiated cells existing in the cell/polymer constructs on the construct osteoinductivity in vivo. Lastly, we will evaluate the ability of optimized cell/polymer constructs created in vitro under flow perfusion culture and loaded with deposited osteoinductive extracellular matrix and secreted angiogenic factors to form new bone in an orthotopic site and restore osseous continuity using a critical size cranial defect model in rats. New bone formation will be assessed radiographically and histomorphometrically as a function of time. Light and fluorescence microscopy will allow quantitative and qualitative analyses of the extent, character, and dynamics of new bone formation. The mechanical properties of the grafted bones will be measured to verify restoration of the integrity of the reconstituted region. This project will provide clinically valuable information regarding new tissue-inducing orthopaedic biomaterials which are becoming particularly important because of the renewed concern for the safety of non-degradable implants and the potential for disease transmission with allografts.